The affinity for binding displayed by antibodies towards cell surfaces is often exploited as the basis for imaging systems used for cytometric and/or hemotologic analysis of cell samples (hereinafter test samples). Imaging systems often employ antibodies as binding molecules which specifically bind sites on the surfaces of specific cells contained in a test sample. In order to detect whether the antibody has bound to the surface of a cell, it is tagged or labeled with a fluorescent molecule. The antibody and its fluorescent molecule are collectively referred to as a conjugate.
In a typical cytometric or hemotologic analysis, the conjugate is contacted with a test sample, which is usually blood or a fraction thereof which contains a variety of cell populations, to form a test mixture. The mixture is incubated for a time and under conditions sufficient for the conjugate to bind target sites on the surface of certain cell populations. After the incubation period, an energy source excites the fluorescent molecule of the conjugate, thereby causing it to fluoresce. This fluorescence is detected using, for instance, a camera that detects cell images via the fluorescence of the bound conjugate. Cameras currently used in imaging systems are highly sensitive and as a result, are very expensive. These cameras are necessarily sensitive because they must detect conjugates that have a relatively low fluorescence. For example, the conjugates currently used in imaging systems typically have a Molecules of Equivalent Soluble Fluorochrome (MESF) value of approximately 12,000. A fluorescently stained cell having a MESF value of 12,000 can reliably be imaged by a photometric cooled Charged-Coupled Device (CCD) camera with 12 bits, 4,096 levels and 500.times.386 pixels. This type of camera costs approximately 20,000 dollars and is a major cost associated with the production of an imaging system.
There have been several attempts to produce a conjugate which has a MESF value that is detectable by a less sensitive and thereby less expensive camera. Previous attempts to enhance the fluorescence of conjugates have sacrificed the conjugates binding efficiency for a brighter conjugate. For example, in an attempt to increase the fluorescence of a conjugate, antibody has been randomly labeled with multiple fluorescent molecules (sometimes referred to as fluorophores). While this random labeling increases the number of fluorophores per antibody, it also binds fluorophores to the binding region of the antibody. When this region is thus bound by a fluorophore, it is incapable of binding its target and thus cannot image cell surfaces and serve its intended purpose. In addition, labeling an antibody with multiple fluorophores often leaves the Fc portion of the antibody unhindered and capable of binding Fc receptors which may be present on the surface of cells contained in a test sample. Because of their ability to bind in this manner, non-specific binding of the conjugate occurs and misleading images are the result.
In another attempt to increase the fluorescence of imaging conjugates, multiple fluorophores have been attached to a polymer and the polymer was attached to an antibody. However, this conjugate does not serve its intended purpose because it suffers significant quenching, and therefore, signal loss caused by the inadequate spacing between multiple fluorophores on a polymeric backbone that has a limited amount of space.
In yet another attempt to increase the fluorescence of imaging conjugates, fluorescent microparticles or colloidal particles have been attached to an antibody thereby increasing the fluorescence of the conjugate. However, this type of conjugate suffers the malady of being insoluble. Because these conjugates are insoluble, they are recognized as foreign bodies by phagocytes that are often present in test samples. As a result, these conjugates are ingested by the phagocytes and the fluorescence associated with such a cell is due to the fluorescent particle in the phagocyte, not the result of a conjugate bound to a marker on the surface of a cell.
Molecules known as cyclodextrins have been used in the art of conjugate synthesis. Cyclodextrin is a well known water soluble cyclic oligosaccharide having a hydrophobic center cavity and a hydrophilic outer region. Generally, the shape of a cyclodextrin molecule is cylindrical with one end of the cylinder having a larger opening than the other. The larger opening is known as the secondary rim and the other opening is known as the primary rim. A cavity into which small molecules can enter through the larger secondary rim is present between the two openings of the cyclodextrin molecule and, in aqueous systems, the cavity of a cyclodextrin molecule (the "host") provides a hydrophobic microenvironment for the complexing of small molecular weight hydrophobic molecules (the"guest").
Efforts to generate polymeric cyclodextrin have also been made in an attempt to increase the fluorescence associated with conjugates. Theoretically, the complexing properties of a single cyclodextrin molecule can be magnified by having several cyclodextrin molecules in close proximity to each other (i.e. having several cyclodextrin molecules in close proximity to each other increases the probability that a guest molecule will enter the cavity of a cyclodextrin molecule). Thus, as the theory goes, if a polymeric cyclodextrin molecule were created, it would be capable of hosting a plurality of guest molecules. Further, if the guest molecules of a polymeric cyclodextrin molecule were signal generating groups, there would be several, for instance, fluorophores in close proximity to each other and the fluorescence associated with the polymer would be greater than that of a single fluorophore. Hence, if a conjugate were made with a fluorophore containing polymeric cyclodextrin its fluorescence would, theoretically, be greater than a conjugate made with a single fluorophore.
Several cyclodextrin based polymers have been manufactured to validate the above mentioned theory. However, these polymers suffer from problems that severely limit their desired effect. These cyclodextrin based polymers are synthesized using cyclodextrin monomers that have been modified to contain several reactive groups on the cyclodextrin's primary and secondary rims which allows these monomers to react via their primary and secondary rims, and react multiple times via their multiple reactive groups. When a cyclodextrin molecule is bound by its secondary rim, the larger opening to the hydrophobic cavity is hindered. As a result, it is difficult for a guest molecule to enter the cavity of the cyclodextrin, and the cyclodextrins utility as a host is sacrificed. Further, forming polymers with cyclodextrins having multiple reactive groups, allows a high degree of crosslinking. When crosslinking occurs not only are the cyclodextrins bound by the secondary rim, causing the problems mentioned above, but a matrix of cyclodextrins forms. Consequently, the number of cyclodextrins polymerized is limited and many of the cyclodextrins polymerized get buried within the matrix. Although many cyclodextrins are in close proximity, very few of them have accessible secondary openings and very few guest/host complexes are able to form. The problems associated with the above polymers stem from their methods of production. Specifically, the monomeric cyclodextrins employed to synthesize the polymers are over-reactive.
In order to synthesize a useful polymeric cyclodextrin it is necessary to have a properly reactive monomeric cyclodextrin building block. An example of such a reactive cyclodextrin is 6-cyclodextrin monoaldehyde. Previous routes to 6-cyclodextrin monoaldehyde have been described, but these synthetic procedures require multiple steps which include the synthesis of toxic and potentially explosive intermediates. Additionally, these procedures require materials that are hard-to-obtain and expensive. Thus, in order to effectively use the complexing properties of cyclodextrin, particularly in relation to conjugate synthesis, a safer and more efficient route to 6-cyclodextrin monoaldehyde is needed.
Reducing the expense of imaging systems can be accomplished by reducing the cost of one of its most expensive components. Specifically, if a low cost camera were able to be used in an imaging system the cost of the entire system would be greatly reduced. Given the present state of imaging conjugate technology this is not practical. There is therefore a need for a conjugate capable of emitting a signal capable of detection by a low cost camera.